Introduction To Histology

Question 1

LO: Define histology

Answer 1

The study of microscopic structure of tissues

WHY STUDY HISTOLOGY?
It is an advantage to be able to identify morphological changes in tissues that characterise disease.
Allows for accurate diagnosis

Question 2

LO: Recognise the significance of microscope resolution in the study of microanatomy

Answer 2

In order to observe and discriminate between tissue and cell components they must be enlarged (magnified)

Cell membrane: ~7-5nm thick
Nucleus: ~ 4um across
Nucleolus: ~ 1um across
Mitochondrion: ~ 0.5 - 2um in length
Cilium: ~250nm across

Question 3

LO: List and explain the basic steps in tissue processing

Answer 3

1. FIXATION: Treating with a chemical reagent (fixative) to preserve tissue architecture
   (A) holds tissue components in normal places during subsequent processing
   (B) prevents tissue from rotting due to bacterial/fungal attack or auto digestion by enzymes leaking from lysosomes
   Fixatives have binding sites enabling them to form cross-links between two adjacent tissue proteins thus fixing them in place
   Most common fixative is formaldehyde
   Limitation: Fixation preserves proteins but not all other important cell components
2. EMBEDDING: Hardening the tissue so that it may be sectioned, most commonly by replacing the water in tissue with paraffin wax or resin
   (A) DEHYDRATION: water is removed by gradually replacing it with alcohol;
   (B) CLEARING: alcohol has to replaced with an organic solvent, such as xylene, which will mix with wax or resin. The tissue goes transparent. The solvent is replaced by hot molten wax (58ºC) which is cooled to harden or a resin solution which is heated to harden (60ºC)
   Limitations: dehydration and heating can damage tissue architecture. Hardening by freezing can result in formation of ice crystals that tear holes in the tissue. Clearing dissolves not only alcohol but tissue lipids as well
3. SECTIONING: Embedded block is cut as thinly as possible using a microtome. Ideally no more than the thickness of a single cell (5-7 um) for LM or of an organelle (~100nm) for EM
4. MOUNTING: Tissue sections then mounted onto glass slides
5. DE-WAXING: Wax must be removed from sections to allow stains to penetrate and bind to tissue components. Reversal of previous steps; sections are dipped in xylene, then alcohol and finally water
6. STAINING: Cells are virtually colourless and must be stained for visualisation under the microscope
   (A) Routine stains: traditional stains which distinguish the nucleus from the cytoplasm for LM
   (B) Special (histochemical) stains: stains which identify specific cellular chemical or enzyme components

Two most common routine stains are:
1. Haematoxylin & eosin (H&E): standard histology/histopathology stain
(A) haematoxylin: a basic dye that binds to nuclei acids, ribosome and rough endoplasmic reticulum due to their high content of DNA and RNA. Stains them purplish-blue
(B) eosin: an acidic dye that binds to both intracellular and extracellular proteins, staining them red

2. Trichrome: a mixture of three dyes; a basic due for nuclei (colour varies)and two acidic dyes for proteins, one for intracellular proteins (usually red-pink) and one for extracellular proteins (usually blue-green)